There are four extant species of horseshoe crabs. The three Asian species, Tachypleus tridentatus, T.gigas and Carcinoscorpius rotundicauda, are dis- tributed in the southeastern region of Asia from Bay of Bengal to the Inland Sea of Japan. The American species, Limulus polyphemus, is found along the eastern coast of North and Central America from Nova Scotia (Canada) to the Yucatan Peninsula (Mexico).Each hemocyanin of the three Asian horseshoe crabs is composed of six immu-nologically different subunits, each subunits of which is immunologically identical with the comparable subunits of two other species. The N-terminal amino acid sequences of these hemocyanin subunits were analyzed and compared one another. From the sequence comparison and immunological comparison of hemocyanin subunits, it is evident that Asian horseshoe crabs share six or- thologous hemocyanin subunits which are immunologically identical.On the other hand, Limulus polyphemus have eight immunologically different hemocyanin subunits and the N-terminal sequences of the eight subunits were already analyzed.From the sequence comparison of the hemocyanin subunits among four horse- shoe crabs, it is inferred that the common ancestor of four horseshoe crabs had six hemocyanin subunits and that, after the divergence of two lineage of L.polyphemus and Asian horseshoe crabs, duplications of two subunits gave rise to two subunit sets in the lineage of L.polyphermus and gave the eight different subunits to L.polyphemus. Thus I concluded that the four extant horseshoe crabs share six orthologous hemocyanin subunits derived from their common ancestor.